Field of Invention
[0001] This invention concerns luminescence imaging methods and apparatus, particularly
methods and apparatus which employ optical fibres and a CCD to inspect and monitor
luminescence from arrays such as microtitre plates.
Background
[0002] Reference is made to Patent Specifications GB 2339900, GB 2339901, and GB 2351555,
which describe an epifluorescence imaging system designed to measure the fluorescence
emitted from the bottom of an array of samples when they are irradiated from beneath
by excitation light. Figure 1, taken from these Applications, shows the use of an
array of typically 8 x 12 trifurcated fibre optic bundles arrayed in a presentation
plate on which the sample plate is placed. One sub-set of fibres within a bundle is
used to bring in the excitation light, and two other sub-sets of fibres in each bundle
collect emitted fluorescent light into two separate filter channels to enable simultaneous
measurement of dual wavelengths. After the filters the light is transmitted through
single fibres, 2 x 96 of them, to a fibre optic input face of a CCD camera. The arrangement
of optical fibre bundles at the filters is shown in Figure 2, and of the optical fibres
at the input to the CCD camera is shown in Figure 3. Figure 4 shows how the different
categories of fibres are arranged in a hexagonal pattern within a bundle.
[0003] Typically an image can be taken every second, and repeated at approximately one-second
intervals.
[0004] This system has proved to be an efficient, highly sensitive measurer of fluorescence
in dual wavelengths, and the ability to measure dual wavelengths simultaneously is
of great value for certain assays, such as FRET assays, where a change in the ratio
of the signal strengths in the two wavelengths is an indicator of chemical binding
or some other attribute of the assay.
[0005] This 96-channel system can be used to measure 96-well microtitre plates, or by stepping
the microtitre plate over the presentation plate, larger assay presentations such
as 384, 864, 1536, 3465...., well plates (i.e. having and n
2*96 pattern, where n is an integer).
[0006] This system is not optimised for the measurement of luminescence for which no excitation
is needed, and in which a sample emits light by virtue of a chemical reaction occurring
when reagents are interacting in the sample well.
[0007] Thus, for fluorescence, the fibre bundle is stood off below the well since light
has to illuminate the sample evenly and receive fluorescence light with uniform efficiency
from all parts of the well. This still applies even if the sample is a cellular layer
on the base of the well.
[0008] Therefore although this epi-fluorescence system can be used to measure luminescence,
it is not optimal, and is generally sensitive enough only for the brightest types
of samples and assays. Examples of such assays include detection using luciferase
enzyme; calcium detection, for example in cells, linked to the enzyme aequorin, which
produces a flash of light during the reaction; and alkaline phosphatase linked assays
using enhanced chemiluminescence substrates.
Summary of the Invention
[0009] According to one aspect of the present invention an imaging system such as has been
proposed for use with epi-fluorescence may be adapted for use with luminescence samples
(which in general produce less light than epi-fluorescence samples) by optimising
the position and type of fibre optic read out.
[0010] In one embodiment of the invention it is proposed to use a second presentation plate
in which, say, 8 x 12 single fibres are mounted, each with its face immediately below
or in contact with the base of the sample plate. The end face of each fibre which
is to view the plate is preferably polished. Typically the fibres are optical fibres
of plastics fibre material, say 2mm diameter, which is available at low cost with
relatively high numerical aperture (NA) - e.g. NA = 0.5. This is to be compared with
the NA (typically about 0.2) of the silica fibres which are commonly used to make
the fibre bundles for an epifluorescence system. Silica fibre material is used to
minimise the auto-fluorescence background that can occur in the presence of excitation
light. The use of plastics material for fibres in fluorescence systems is ruled out
since plastics materials tend to fluoresce.
[0011] Using such an arrangement of plastics fibres, a factor of light gathering improvement
of the order of 10-20 has been obtained for luminescence samples when compared with
the measurement of these samples using an epi-fluorescence system. This factor results
both from the closer placing of the end of each fibre to the sample, and from the
higher NA of the fibre. This makes possible the measurement of weaker types of luminescence.
[0012] According to a second aspect of the invention an epi-fluorescence imaging system
may be adapted for use with luminescence samples by leading a second set of high NA
fibres to the input of the CCD to which the fibres employed for epi-fluorescence samples
are also led, the two sets of fibres being arranged interstitially, so that a second
CCD camera is not needed.
[0013] Although the combined system will be used either for fluorescence or for luminescence,
there is no risk of cross talk between the closely spaced fibres, since only one set
at a time is illuminated.
[0014] According to a further aspect of the invention the luminescence light can be analysed
in two wavelengths simultaneously by means of a bifurcation of each of the fibres
employed when looking for luminescence. Thereafter there would be two parallel filter
channels, and the two sets of fibres would be mounted interstitially to the two sets
of fluorescence transfer fibres.
[0015] Certain types of luminescence assays benefit from analysis at two wavelengths. Examples
of this include multi-labelled assay systems, where more than one label has been used
in an assay. A further example is resonant energy transfer type assays, where a donor
molecule emits light at one wavelength and a receptor molecule emits light at a second
wavelength. The degree of energy transfer between the two molecules is measured by
the ratio of the intensity of the two wavelengths emitted by the molecules.
[0016] The analysis algorithms already proposed (such as referred to in the Patent Specifications
listed earlier) for performing measurements on fluorescence derived signals can be
readily extended to luminescence signals from the CCD camera. Thus for example in
the case of a 96-fibre array, a set of geometrically fixed 96 spots of light (or 2
x 96 spots) can be registered at a fixed displacement on the CCD from the 2 x 96 fluorescence
spots.
[0017] A fixed array of fibres mounted in a presentation plate for luminescence measurements
can be used independently of the epifluorescence system. An example would be a 16
x 24 fibre array to measure 384 channels simultaneously.
[0018] The fibres in the filter region may be arranged on a 100mm-diameter filter and the
system would require its own CCD camera, but this would be the only significant additional
element.
[0019] The 384 fibres at the input to the CCD camera may be re-arranged if required to accommodate
the aspect ratio of the CCD.
[0020] Such an arrangement can also measure a 1536 well, plate by means of a 2 x 2 stepping
of the sample plate relative to the presentation plate.
[0021] As before, each fibre can be bifurcated to enable dual wavelength analysis. This
system represents a low-cost, optically efficient means of measuring luminescence
in larger size well plates, (384, 1536 wells or more).
[0022] In an arrangement embodying the invention for luminescence samples, a fixed fibre
array for the measurement of arrays of samples has the following advantages over a
lens based method.
(1) absence of parallax (so that wells in the corners of a plate are measured as efficiently
as those in the centre of the plate) which is only avoided by using an (expensive)
telecentric lens;
(2) no vignetting;
(3) no need to focus:
(4) there is much more efficient light gathering than from a lens.
[0023] Thus an optical fibre placed immediately under a well of about the same diameter
has optical efficiency approximately equal to the (NA)
2 =(0.5)
2 =0.25. On the other hand a quality telecentric lens (say F=1) imaging a whole plate
(size 110 x 75mm) onto a CCD (size 25 x 25 mm) so that the lens demagnification is
m=110/25 = 4.4, has optical efficiency 1/{2F(1 +m)}
2 = 0.0086.
[0024] A set of optical fibres as proposed is therefore about 30 times more efficient, and
much less expensive than a quality lens.
[0025] The advantages of the invention are achieved if the end of each fibre that is to
collect light from a sample is very close to, if not touching, the sample, either
above or below the sample, such as a well in a multi-well plate, for example.
[0026] The invention also lies in a system for inspecting light emitting samples such as
are contained in a well plate from each of which at least two optical fibres or bundles
of fibres lead to a CCD camera, the fibres or bundles of fibres from all samples being
arranged in two sets, a first set which are formed from a non-fluorescing material
and a second set which are formed from a material which may fluoresce but enables
the fibres formed therefrom to have a higher numerical aperture than those of the
first set, one of the said at least two fibres linked to each sample belonging to
the one set, and the other to the other set, wherein the fibres are arranged interstitially
at the input to the camera, the light emitted from each fibre falling on a uniquely
addressable region of the CCD, and the addressing of the CCD is arranged so that the
addresses associated with one set of fibres, or the other, are read out and refreshed
to produce an output signal for analysis.
[0027] By reading out the addresses relating to the first set of fibres, an output signal
is obtained relating to any fluorescence from the samples.
[0028] By reading out the addresses relating to the second set of fibres an output signal
will relate to any luminescence produced by the samples.
[0029] Where the samples need to be excited so as to produce fluorescence, additional fibres
lead to the samples from an excitation source.
[0030] Additional sets of fibres, each set illuminating a different unique set of addresses
of the CCD, may be provided with appropriate wavelength selection as by filtering,
to enable dual wavelength analysis to be performed on light emitting samples.
[0031] Using bi-furcated fibres in the second set, so as to produce 2N fibres leading from
N wells, with one set of N leading to the CCD input, may allow the other set to be
used for transmitting excitation radiation from a suitable source to the samples,
thereby obviating the need for a third set of N fibres to the wells, for conveying
excitation radiation thereto.
[0032] The invention is illustrated by way of example in Figs 5 onwards.
[0033] The invention extends the current epi-fluorescence system into a whole new application,
namely detection of luminescence, at a minimal cost, and in a technically simple manner.
As before, microtitre plates having 384, 864, 1536.... wells, based on a 96-channel
pattern, can be measures by stepping the sample plate over the presentation plate.
Figure 5 shows a 96-channel luminescence reading head.
[0034] A filter can be added to this luminescence system, as shown in Figure 7, which needs
the same arrangement of 96 extra fibres at the CCD shown in Figure 6.
[0035] The arrangement of fibres in the filter space would be as in Figure 2. As with the
fluorescence version, there is a rearrangement of the fibres from the sample presentation
plate to the filter space, if a filter is used, and again to the input to the CCD
camera. Note that the filter space does not necessarily need to be as thick as in
the fluorescence version, as there is no excitation light that has to be blocked from
entering the emission path, allowing simpler, thinner filters to be used for the luminescence
filter space.
[0036] Fig 6 shows the interstitial arrangement of the fibres to be used for transmitting
luminescence light to the camera, with those of the known epi-fluorescence system.
All the fibres lead to the one entrance to the CCD camera.
[0037] Bifurcation of the luminescence fibres is shown in Fig 8.
[0038] Interstitial placement of the two sets of fibres is again shown in Fig 9.
[0039] Fig 10 shows an example of a 16 x 24 fibre array, which allows for simultaneous inspection
of all the wells in a 384 well plate. As shown in Fig 11, the 384 fibres are arranged
on a 100mm diameter filter where they extend through the filter space of the epifluorescence
system.
[0040] Fig 12 shows the arrangement of the 384 fibres at the CCD camera input.
1. A system for inspecting light emitting samples such as are contained in wells in a
well plate, characterised in that at least two optical fibres lead to a CCD camera from each sample that is being inspected,
the fibres comprising two sets, a first set which are formed from a non-fluorescing
material and a second set which are formed from a material which may fluoresce but
have a higher numerical aperture than those of the first set, one of the said at least
two fibres linked to each sample belonging to the one set, and the other to the other
set, and wherein the fibres are arranged interstitially at the input to the camera,
the light emitted from each fibre falling on a uniquely addressable region of the
CCD, and the addressing of the CCD is arranged so that the addresses associated with
one set of fibres are read out and refreshed separately from those associated with
the other set of fibres, to produce separate output signals for analysis.
2. A system according to claim 1, wherein, by reading out the addresses relating to the
first set of fibres, an output signal is obtained relating to any fluorescence from
the samples, and by reading out the addresses relating to the second set of fibres
an output signal is obtained relating to any luminescence produced by the samples.
3. A system according to claim 1 or claim 2, wherein an additional fibre leads to each
sample from an excitation source, which is operated when the samples need to be excited
so as to produce fluorescence.
4. A system according to claim 3 wherein bi-furcated fibres are employed in one set of
fibres, one of each of which is used for transmitting excitation radiation from a
suitable excitation source to a sample, and the other for conveying light from the
sample to a region of the CCD.
5. A system according to any of claims 1 to 4, wherein an additional set of fibres extends
between the sample and the CCD camera, one from each well, and each of the additional
set of fibres illuminates a different unique address in the CCD, and a filter is provided
in the path of the fibres in the additional set, to enable wavelength analysis to
be performed on light emitted from the samples.